1. Field of the Invention
The present invention relates to a mutant tyrosine repressor that does not require tyrosine to induce expression of a tyrosine phenol-lyase gene. The present invention also relates to a gene encoding the mutant tyrosine repressor, and utilization thereof. The mutant tyrosine repressor is useful in the field of fermentation, for example, in the production of L-3,4-dihydroxyphenylalanine, hereinafter, “L-DOPA”.
2. Brief Description of the Related Art
L-DOPA is a precursor of dopamine. Dopamine is a nerve transmitter and is useful as a therapeutic agent for Parkinson's disease, and so forth. L-DOPA has conventionally been manufactured by chemical synthesis. In recent years, however, L-DOPA is enzymatically synthesized from catechol and serine, or catechol, pyruvic acid, and ammonia using tyrosine phenol-lyase (TPL).
TPL is known to be produced by a wide variety of microorganisms, including those belonging to the genus Escherichia, Pseudomonas, Flavobacterium, Bacillus, Serratia, Xanthomonas, Agrobacterium, Achromobacter, Aerobacter, Erwinia, Proteus, Salmonella, Citrobacter, Enterobacter, Pantoea, or the like (Japanese Patent No. 2521945 or Japanese Patent Publication (Kokoku) No. 6-98003). Enzymatic characteristics of TPL have also been elucidated (Biochem. Biophys. Res. Commun., 33, 10 (1963)). Furthermore, nucleotide sequences of the structural gene and an upstream region of the TPL gene from Erwinia bacteria, which highly expresses TPL, have been reported (Suzuki, H. et al., J. Ferment. Bioeng., 75, No. 2, 145-148 (1993)).
It is known that expression of TPL is regulated by a tyrosine repressor. That is, the tyrosine repressor is activated when tyrosine binds to it, and thereby expression of the TPL gene is induced. The structure of the tyrosine repressor gene has been elucidated for Escherichia coli (Cornish, E. C. et al., J. Biol. Chem., 261, 403-410 (1986)). Furthermore, the inventors of the present invention successfully isolated a gene encoding a tyrosine repressor from Erwinia bacteria and reported that the tyrosine repressor has an activity of positively regulating expression of the TPL gene (U.S. Pat. No. 6,146,878).
Production of L-DOPA using microbial cells has also been attempted. To efficiently produce L-DOPA, it is important to use a microorganism which expresses a high amount of TPL. The addition of large amounts of tyrosine to a culture medium is required since tyrosine induces the expression of TPL. However, the extremely low solubility of tyrosine in water causes a large amount of tyrosine, which can contaminate the L-DOPA reaction system during cultivation, resulting in difficulty purifying and isolating L-DOPA. To solve this problem, a mutant tyrosine repressor that is able to express a high amount of TPL in the presence of small amounts of tyrosine has been developed (Japanese Patent Laid-open No. 2001-238678). However, this mutant expresses TPL more efficiently when tyrosine is added, and therefore development of a microorganism that expresses large amounts of TPL in the absence of tyrosine is desired.
The microorganisms disclosed as Erwinia herbicola in U.S. Pat. No. 6,146,878 and Japanese Patent Laid-open No. 2001-238678 and so forth have been reclassified into Pantoea agglomerans. Therefore, the aforementioned Erwinia herbicola will be referred to as Pantoea agglomerans hereinafter.